Project Summary/Abstract Selenium is an essential trace element long known for its antioxidant properties, most or all of which are attributable to selenoproteins. The known functions of selenoenzymes include protecting cell membranes, proteins and nucleic acids from cumulative oxidative damage. Selenium is incorporated cotranslationally into proteins as the amino acid, selenocysteine, and is specified by UGA, which typically functions as a stop codon. This occurs via secondary structures in the 3'UTRs of eukaryotic mRNAs, termed SECIS elements, and the factors they recruit. Delineation of these elements by the PI of this application provided the ligand for identification of SECIS binding proteins, including SBP2, ribosomal protein L30, and nucleolin. These signature sequences also formed the basis for bioinformatic approaches to identify all of the selenoprotein genes in completely sequenced genomes, resulting in identification of 25 selenoproteins in humans and 24 in mice. Selenocysteine is synthesized on a specialized tRNA, utilizing a phosphoserine precursor and selenophosphate as selenium donor. Extensive efforts from a small number of laboratories have resulted in identification of many of the factors involved in selenocysteine biosynthesis and its cotranslational insertion into proteins. Accumulating evidence indicates that the processes of selenocysteine biosynthesis and incorporation into proteins occur through a series of interacting proteins that form complexes, increasing the efficiency of incorporation and recycling of the necessary factors Additional insights into the mechanisms by which selenoprotein mRNAs elude nonsense mediated decay are being unveiled through studies of the interactions among these factors and their subcellular localization. The overall goals of this proposal are to continue our investigation of the mechanism of selenocysteine incorporation, and the mechanisms by which selenoprotein mRNAs elude nonsense mediated decay, also termed mRNA surveillance. This will be accomplished via three new specific aims that employ a tandem mass spectrometry proteomics approach. The first aim is identification of factors that interact with selenoprotein mRNAs in vivo using a novel RNA-protein affinity purification strategy. The second aim will use tandem affinity purification of protein complexes to identify all of the components of the selenoprotein biosynthesis machinery. Using this same approach, we propose to identify factors conferring differential sensitivity of selenoprotein mRNAs to nonsense mediated decay, an mRNA surveillance mechanism thought to play a key regulatory role in dictating which selenoproteins are synthesized when selenium is limiting. The third aim will investigate the sequential assembly of factors into the complexes that mediate selenoprotein synthesis. Through these studies, we hope to gain insights into the mechanisms by which organisms have evolved to maximize utilization of this essential trace element in development and disease prevention.